THE JOURNALOF BIOLOGICAL CHEMISTRY Vol. 259, No. 2, Issue of January 25, pp.

1299-1308, 1984
The American Society of Biological Chemists, Inc.
(5 1984 by Prmted in Li S.A.

Purification andCharacterization of the Lactate Dehydrogenase

(LDH-B4) Allozymes of Fundulus heterocZitus*
(Received for publication, July 26, 1983)

Allen R. Place$ and Dennis A. Powers4

From the Departmentof Biology, The Johns Hopkins University, Baltimore, Maryand21218

In order to evaluate whether structural differences
exist between allelic variants of a B-type lactate de-
hydrogenase (LDH; L-lactate:NAD+ oxidoreductase,
EC 1.1.1.27) in theminnow Fundulus heteroclitus, the
allozymes (LDH-B:, LDH-Ba/Bb, and LDH-B:) were
purified to homogeneity by affinity chromatography.
Each variant was characterized as to holoenzyme and
subunit molecular mass, isoelectric point (PI), thermal
and urea stability, and susceptibility to proteolysis.
Differences in electrophoretic mobilities were due to a
lower PI for LDH-B: (PI = 6.6) than for LDH-Bt (PI =
heteroclitus. Natural populations of this fishpossess three
electrophoretically distinguishable phenotypes (LDH-B!,
LDH-Ba/Bb,and LDH-B;) which segregate autosomally (6).
A dramatic latitudinal change in gene frequency exists for
this polymorphismalong the Atlantic coast of the United
States (7). Populations from Maineare greater than 90%
homozygous for the Bballele, whereaspopulations from Geor-
gia and Florida are greater than 94% homozygous for the B”
allele. Since the east coast has one of the steepest thermal
gradients in the world (7) and because temperature has a
profound effect on protein structure and function in cold-

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7.2). Stability to inactivation by heat, urea, and pro-
teolysis was in each case: LDH-B: > LDH-B’/Bb >
LDH-B;. Inactivation by trypsin may involve the ar-
ginine-richcatalytic loop of lactate dehydrogenase
(50). The results suggest that the allozymes differ in
their conformational flexibility.
blooded organisms like fish, we asked whether changes in
gene frequency might result from a selective adaptation to
different thermal environments. The present two papers pro-
vide evidence consistent with such an adaptation.
EXPERIMENTAL PROCEDURES

Materials
Approximately one-half of allgenescodingfor proteins Chemicals
have naturallyoccurringvariants,that is, theyare poly- The materials used were obtained from the following sources:
morphic (1, 2). The evolutionaryforces responsible for main- sodium pyruvate,lithium ( L - ( + ) ) lactate,phenazinemethosulfate,
taining this genetic diversity remain controversial (3-5). Pro- nitroblue tetrazolium, NADH, NAD+, 2-mercaptoethanol, BSA? and
Tris from Sigma; phenylglyoxal and dithiothreitol from Aldrich; SDS
ponents of one hypothesis, the selectionists,advocate a form from Matheson, Coleman, and Bell; Coomassie brilliant blue R-250,
of natural selection to maintain proteinpolymorphisms while acrylamide, N,N’-bismethylene acrylamide, and TEMEDfrom East-
exponents of the other hypothesis, the neutralists, argue thatman-Kodak Organic Chemicals; urea, ferritin (equine, amorphous
most allozymes are effectively immune or neutral to natural 5x-crystallized), and ammonium sulfate (enzyme grade) from
selection. Implicit in the neutralist hypothesis is that most Schwarz/Mann; Sephadex G-200, protein standards (Lot CH31; al-
structural differences among allozymes are, in essence, func- dolase, ovalbumin, chymotrypsinogen, and ribonuclease A), and blue
dextran 2000 from Pharmacia;ultrafiltration membranes (PM-10
tionally equivalent (4). and PM-30) from Amicon; carrier ampholytes, pH 3.5 to 10 (3/76;
As one test of this hypothesis, we undertook a detailed Batch No. 39) andpH 5 to 8 (11/75; Batch No. 16) from LKB
investigation of the structural and functional properties of an Produkter AB. Other chemicals used were of analytical grade and
LDH-B4’polymorphism in the near shoreminnow, Fundulus purchased from local suppliers.
- Enzymes were obtained from the following sources: chymotrypsin
* These studies were supported by Grants DEB76-19877, DEB79- (CDI 365771) and trypsin (L-1-tosylamido-2-phenylethyl chlorome-
12216, and DEB82-07006 from the National Science Foundation. The thy1 ketone-treated; TRTPCK 36H724) from Worthington; pronase
material presented in this paper is part of the thesis submittedby A. (B grade) from Calbiochem; soybean trypsin inhibitor, bovine heart
R. P. to the Department of Biology, The Johns Hopkins University, lactate dehydrogenase (LDH-B,, 99%),and rabbit muscle lactate
Baltimore, MD in partial fulfillment of the requirementsfor the dehydrogenase (LDH-A,) from Sigma.
degree of Doctor of Philosophy. A preliminary report of the data in All buffers and reagents were made with deionized glass-distilled
this paper was presented at the61st Annual Meeting of the Federation water.
of American Societies for Experimental Biology (Fed. Proc. (1977)
36, 738). This is Contribution No. 1227 from the Department of Organisms
Biology. The costs of publication of this article were defrayed in part Adult fish were caught andtheir livers dissected as described
by the paymentof page charges. This article must therefore be hereby elsewhere (6).Three localities were sampled: Wiscasset, ME; Annap-
marked “aduertisement” in accordance with 18 U.S.C. Section 1734
solely to indicate this fact. used in describing theLDH-B4 allelic variants of F. heteroclitus
$ Supported by Training Grant HD00139 from the National Insti- corresponds to theirrelative electrophoretic mobility at pH7.00. The
tutes of HealthtotheDepartment of Biology. Present address: faster in mobility is given the superscript a, whereas superscript b is
Department of Biology, University of Pennsylvania, Philadelphia, used for the slower in mobility. Subscripts willbe reserved for
P A 19104. chemical designations such as subunit composition. LDH-B”/Bb re-
I To whom correspondence should be addressed. fers collectively to all five forms of lactate dehydrogenase produced
In keeping with the IUPAC-IUB recommendations on the no- in individuals heterozygous at thislocus.
menclature for multiple forms of enzymes (1977, J . Biol. Chem. 252, ‘The abbreviations used are: BSA, bovine serum albumin;
5939-5941), the muscle- and heart-type lactate dehydrogenases are TEMED, N,N,N‘,N,-tetramethylethylenediamine; SDS, sodium do-
designated as LDH-A, and LDH-B,, respectively. The nomenclature decyl sulfate; N6-AMP, N6-(6-aminohexyl)-AMP.

1299
1300 Allozymes
Dehydrogenase Lactate
olis, MD; and Sapelo Island,GA. Individuals from Maine and Georgia
were used as sources for the LDH-B! and LDH-B; allozymes, respec-
tively. Individuals from Maryland were screened for their LDH-B4
phenotype by the fin-cliptechnique described previously (6) and
tissue from each phenotype was pooled.
Preparation of Affinity Gels
The two affinants used were synthesized en bloc prior to coupling
to Sepharose 4B. N6-AMP was synthesized according to Mosbach (9)
from the 6-chloropurine riboside, while N-(6-aminohexyl)oxamic acid
was prepared as described (10). The conditions for cyanogen bromide
activation are as published (10). Typically, 4-6 @molof N6-AMP/ml
of gel and 10-11 pmol of N-(6-aminohexyl)oxamate/mlof gel were
coupled.
Methods
Purification of F. heteroclitus LDH-B, Allozymes
All procedures were performed at 0-4 "C. Unless indicated other-
wise, all buffers and solutions contained 1 mM EDTA (disodium salt)
and 5 mM 2-mercaptoethanol or dithiothreitol. The purified enzymes
were stored as ammonium sulfate suspensions at 4 "C.
Steps I and 11: Extraction and Ammonium Sulfate Fractiomtion-
Frozen livers, 50 g, were homogenized in 250 ml of 0.1 M sodium
phosphate buffer (pH 7.00) with TenBroeck homogenizers. The ex-
tract was centrifuged at 10,000 X g for 30 min and theresulting pellet
was discarded. The supernatant is taken as Step I material.
Solid ammonium sulfate was added to give 30% saturation (4 "C)
and the suspension was stirred for 1h. After centrifugation at 20,000
X g for 30 min, the supernatant was removed and brought to 70%
saturation. Thepellet was discarded. Again the suspension was stirred
and, after 1 h, centrifuged at 20,000 X g for 30 min. The pellet was
redissolved in 20 mM sodium phosphate buffer (pH 7.00) and dialyzed
overnight against four 1-liter changes of this buffer. The resuspended
and dialyzed pellet, after centrifugation at 20,000 X g for 30 min, is
taken as Step I1 material.
Step III: Affinity Chromatography on N'-AMP-Sepharose-To the
Step I1 material, solid NaCl was added to give a 0.2 M solution. The
sample was applied to a column (2.5 X 20 cm) of N'-AMP-Sepharose
which was equilibrated with 20 mM sodium phosphate buffer (pH
7.00) containing 0.2 M NaC1. Absorbance at 280 nm was monitored
until Azso was near zero and then a pulse (20 ml) of 0.25 mM NADH
in the column buffer was applied and elution was resumed. Fractions
containing greater than 90% of the applied activity were pooled as
Step 111 material.
Step IV: Affinity Chromatography on N-(6-Aminohexyl)oxamate-
Sepharose-The pooled fractions (Step 111 material) were applied
directly to a column (6 X 14 cm) of N-(6-aminohexyl)oxamate-
Sepharose (400 ml) equilibrated with 20 mM sodium phosphate buffer
(pH 7.00) containing 0.2 mM NADH and 0.5 M NaCl. Elution was
monitored at 280 nm, correcting for absorbance by NADH in the
elution buffer. Removal of the reduced cofactor from the column
buffer resulted in elution of lactate dehydrogenase as a broad peak.
After concentration by ultrafiltration, the pooled fractions were di-
alyzed overnightagainst 0.1 M sodium phosphate (pH 7.00). The
pooled, dialyzed fractions are taken as Step IV material.
Electrophoretic Procedures
Polyacrylamide Gel Electrophoresis-Disc gel electrophoresis was
performed (11)without sample and spacer gels. Samples, 10 to 50 pl
in 20% glycerol (v/v), were layered under buffer on the upper, cathodal
gel surface. Determinations of size and charge isomers (12) and of
molecular mass were performed in four different concentrations of
acrylamide (4, 5, 6, and 7%). The weight ratio of acrylamide to
bisacrylamide was kept constant at 30 to 0.8. The gel buffer was 0.06
M Tris-HC1, pH 8.0, and the electrode buffer was0.038 M Tris-
glycine, pH 8.3 (11).The samples were dialyzed overnight (4"C)
against 0.083 M Tris-phosphate (pH 6.9) containing 20% glycerol (v/
v) and 5 mM 2-mercaptoethanol. Electrophoresis was performed at 2
mA/tube for 4 h at room temperature (22 "C). Thegels were stained
for either lactate dehydrogenase activity(13) or for proteinwith
Coomassie brilliant blue R-250 (14).3In the latterprocedure, the gels
Both the general protein and lactatedehydrogenase activity stain
were linearly proportional to the quantity of enzyme on a gel only
over a narrow concentration range. The linear range for the general
protein stain, Coomassie brilliant blue R-250, was found to be from
were destained by diffusion against isopropano1:acetic acidH20
(1:1:8). Mobilities, obtained from densitometer tracings at 550 nm,
were calculated relative to the bromphenol blue tracking dye. The
observed RF values were treated according to methods of Rodbard
and Chrambach (15):
Sodium Dodecyl Sulfate-Gel Electrophoresis-Electrophoresis in
gels containing SDS was performed in a slab gel apparatus (Bio-Rad
Model 210) and in tubes (0.6 X 10 cm) utilizing the discontinuous
system described by Laemmli (16). Subunit molecular mass analysis
(17,18) was performed with 10 and 12.5% acrylamide gels; for analysis
of peptides generated by trypsinolysis, 12.5 and 15% acrylamide gels
were used. The following protein standards were employed BSA,
yeast alcohol dehydrogenase, soybean trypsin inhibitor, ribonuclease,
and 0-lactoglobulin.
Isoelectric Focusing
Analytical isoelectric focusing was performed in vertical slab gels
and in tubes (0.6 X 15 cm) according to the procedure of Wrigley
(19). Focusing was conducted at 4 "C for 4 h. The pH gradient was
reconstructed from the measured pH on 1.0-mm sections of gel which
had been eluted for 3 days in 1 ml of deionized distilled HzO. Bovine
LDH-Ba (PI = 6.00) and equine myoglobin (PI = 6.8 and 7.3) were
used as internal standards. The allozymes were detected on the gels
as described earlier, except that prior to stainingwith Coomassie blue
the carrier ampholytes were removed by repeated washes in 12.0%
(w/v) trichloroacetic acid (20).
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Because of the presence ofLDH-B:B" and LDH-BiBf in some
preparations of LDH-Bi, preparative isoelectric focusing was used for
final purification. Focusing (21) was performed with 1%(w/v) carrier
ampholytes, pH 3.5 to 10.0, in a 110 ml LKB 8101-1 electrofocusing
column. The electrofocusing medium contained 5 mM dithiothreitol.
The focusing was performed at 4 "C for 48-72 h at 1 mA, 600 V.
Fractions of 2 ml were collected at the endof the run and the pHof
the fractions was measured. Absorbance at 280 nm and enzyme
activity were determined. The pooled fractions were dialyzed against
0.1 M sodium phosphate (pH 7.00) containing 0.5 M NaCl for 48 h
(4 "C) and the lactate dehydrogenase precipitated with ammonium
sulfate to remove the carrier ampholytes (21). All further character-
ization of LDH-Bq was done with this preparation.
Gel Filtration
Small zone elution (22) analysis was performed on a Sephadex G-
200 column (2.5 X 61 cm) at 4 "C. Elution was conducted in an
ascendingfashion with the flow rate (19.2 ml/h) controlled by a
peristaltic pump. Samples were applied at one-half the elution rate.
The sample was followed with an equal volume of buffer containing
10% (v/v) glycerol. The buffer used throughout was 0.05 M Tris-HC1
(pH 8.00) containing 10 mM EDTA (Na+) and 1 mM dithiothreitol.
The fraction size was 2 ml. Calibration was accomplished using the
following proteins, all at an initial concentration of 20 mg/ml: aldol-
ase, ovalbumin, chymotrypsinogen,and ribonuclease A. Blue dextran
was used to determine the void volume and glycylglycine the internal
volume. The column was calibrated according to the methods of both
Andrews (23) and Ackers (24).
Ultracentrifugation
Sucrose Density Centrifugation-An initial estimate of the sedi-
mentation coefficient was made by sucrose density gradient centrif-
ugation (25). The linear gradients (4.6 ml) used were 15.5 to 33% (w/
v) sucrose in 20mM sodium phosphate (pH 7.2) containing 1 mM
dithiothreitol. Centrifugation was at 4 "C for 17 h at 40,000 rpm in a
SW 50.1 rotor of a Beckman Model L ultracentrifuge. After centrif-
ugation, the gradient was eluted from the bottom of the tube and
0.22-ml fractions were collected. The sedimentation coefficients of
the standard proteinswere those given by Smith (26).
Sedimentation Velocity Ultracentrifugation-In order to permit a
0 to 20pgof lactate dehydrogenase protein, with a lower detection
limit of 0.5 g g . Both homotetramers, L D H - E and LDH-B!!, showed
the same dye-binding efficiency. The tetrazolium stain for lactate
dehydrogenase activity exhibited a more restricted range. The linear
range extended from 0 to 0.13 unit, or approximately 0 to 0.3 pgof
lactate dehydrogenase.
The migration of a protein in polyacrylamide gel electrophoresis
can be described as (18) RF = YO where RF is the mobility of
the protein relative to the front, Yo is the extrapolated RF when T =
0 , KR is the frictional retardation coefficient, and T is the percentage
of acrylamide.
 Dehydrogenase Lactate
direct comparison of the sedimentation behavior of the allozymes,
they were centrifuged in a Beckman Model E ultracentrifuge in pairs
employing two single sector cells with standard 12-mm center pieces
with plane and +lowedge quartz windows. Apparent sedimentation
coefficients were calculated from the rate of boundary migration and
corrected to standard conditions (27). Centrifugation was performed
in 0.1 M sodium phosphate (pH 7.00) a t 20 "C and a rotor speed of
56,000 rpm.
Extinction Coefficient Determination
The extinction coefficients at 280 nm were obtained relative to the
optical density at 280 nm with known protein concentrations deter-
mined by the microbiuret assay. Rabbit muscle LDH-A, (E%= 12.6)
and bovine heart LDH-B, (E% = 14.5) were used as standards.
Assay of Enzymes
For routine assay of lactate dehydrogenase activity, the oxidation
of NADH was followed by observing the decrease in absorbance at
340 nm as function of time. The reaction mixture (3 ml) contained
sodiumphosphate (0.1M, pH 7.5), sodium pyruvate (0.33 mM), NADH
(0.17 mM), and enough enzyme to give an absorbance change of
between 0.1 and O.Z/min. Temperature was maintained at 25 _t 0.1 "C.
One unit of enzyme is defined as that amountwhich causes an initial
rate of oxidation of 1 pmol of NADH/min at 25 "C (1 unit = 16.67
nanokatals).
Allozymes 1301
raised to 37 "C. An aliquot containing 10 pg of lactate dehydrogenase
protein was removed a t each assay time and placed in 25 pl of 0.065
M Tris-HC1 buffer (pH 6.9) containing 10% (v/v) glycerol, 5% (v/v)
2-mercaptoethanol, and3% (w/v) SDS. The sample was heated
immediately in a boiling water bath for 3 min, electrophoresis was
conducted, and the gel was stained for protein with Coomassie blue.
Phenylglyozal Inactiuation-Inhibition with phenylglyoxal (31)
was performed in 0.1 M sodium phosphate buffer (pH 8.00) at 37 "C.
Enzymes at subunitconcentrations of 1.0 X M were incubated
in the presence of a 400 M excess of phenylglyoxal. Aliquots were
removed at various times and assayed for residual activity.
RESULTS
Purification of the LDH-B4Allozymes
Table I summarizes the purification procedure for a prep-
aration of LDH-Ba/Bb. Fig. 1 presents representative chro-
matograms from the two affinity purification steps. The pro-
cedure has been successful in 12 preparations yielding lactate
dehydrogenase with specific activities between 420 and 490
unitslmg. The purification needed from liver to obtain this
purity rangedbetween125- and 300-fold. The addition of
EDTA and dithiothreitol was found to increase stability of
the enzymes in dilute solution. Storage at 4 "C as an ammo-

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Protein Determination nium sulfate suspension retained full enzymatic activity for
Protein concentration was determined by the microbiuret assay periods greater than 8 months.
(28) with BSA and bovine LDH-B, as primary standards.
Criteria for Purity
Stability Studies
The following criteria were sought to establish the purity
Thermal Stability Studies-All thermal inactivation studies were of each preparation: 1) homogeneity on the basis of charge as
performed in 10 mM sodium phosphate buffer (pH 7.50) containing
0.1% (w/v) BSA and 0.15 M NaC1. Purified enzyme was diluted 100- assessed by electrophoresis and isoelectric focusing; 2) ho-
fold to a concentration of 4.6 X lo-' M; aliquots (50 p l ) were placedmogeneity by molecular size and mass as assessed by electro-
in 6 X 50 mm culture tubes and incubated in a water bath at the phoretic and gel filtration and sedimentationvelocity behav-
desired temperature. At time intervals of 5, 10, 20, 40, and 60 min, ior; and 3) constancy of specific activity upon further frac-
triplicate samples were removed and placed in an ice bath. Lactate tionation.Purity of 95% orgreater was obtained for all
dehydrogenase activity was assayed immediately and compared to a preparations.
control sample incubatedat 0 "C for identical times. Fractional activ-
ities remaining (ratio of experimental to control) were averaged for
each time point and analyzed as a first order decay process. From the Characterization of the LDH-B, Allozymes from
temperature dependence of the first orderrate constants, the enthalpy F. heteroclitus
(AH+), entropy ( A S + ) , and free energy (AG') of activation were Table I1 presents a summary of the physiochemical prop-
calculated (29).
Urea Znactiuation-Urea inactivation studies were performed at
erties for the allozymes. According to all the biochemical and
25 "C in 0.1 M Tris-HCI (pH 7.00) containing 1 mM dithiothreitol. physiochemical criteria used, the homologous homotetramers
Duplicate sampleswere incubated in various concentrations of freshly were identical (i.e. LDH-Bt, whether isolatedfrom Mary-
made urea solutions for 10 min in the absence and presence of 0.167 land's Chesapeake Bay or Maine individuals was identical;
mM NADH. The reaction was started by adding pyruvate and NADH similarly, LDH-B; isolated from fish caught in the Chesa-
where necessary to final concentrations of 0.5 and 0.167 mM, respec- peake Bay orGeorgia marshes was identical).
tively.
TO studythe kinetics of the ureainactivation, the incubation Electrophoretic Behuior-From the observed RF values at
mixture contained 0.1 M sodium phosphate (pH 7.00), BSA (1 mg/ each gel concentration and the computer analysis of Rodbard
ml), lactate dehydrogenase (sufficient to give an initial activity of
0.05 unit/ml), and freshly made urea at the specified concentrations.
Incubations were done in 100-ml volumes at 25 k 0.1 "C. At specified TABLEI
time intervals, 2.9-ml aliquots were removed and placed in 3.5-ml Purification summary of allelic B, lactate dehydrogenases
cuvettes. A 0.1-ml aliquot containing 5.0 mmol of NADH and 15
mmol of pyruvate was added to the cuvettes to give final concentra- vel- Total Spe-
Steps pro- cidc Yield dcation
Puri-
tions of 0.167 mM NADH and 0.5 mM pyruvate. ume
tein activitv
- "
Proteolysis Susceptibility-Each allozyme was diluted in 0.1 M "

sodium phosphate buffer (pH 7.00) to a final concentration of 10 pg/ units/

ml. Proteolysis was started by adding each protease (50 pg) to dupli- ml mg mg % -foM
protein
cate samples of the allozymes. After selected time intervals of incu-
bation at 25 "C, aliquots were removed and assayed in duplicate for I. Extraction" 265 6051 1.57 100 1
lactate dehydrogenase activity. Trypsin and chymotrypsin were dis- 11. Ammonium sulfate 68 3253 94.2 2.76 1.8
solved in 1 mM HC1 a t aconcentration of 1 mg/ml; the pronase fractionation
solution (1 mg/ml) was made in 0.1 M sodium phosphate (pH 7.00). 111. Affinity chromatog- 140 35.2 160 102
91.2
The kinetics of inactivation by trypsin was investigated at 25 "C raphy on N6-AMP-
in 0.1 M sodium phosphate (pH 7.50) withatrypsin tolactate Sepharose
dehydrogenase ratio of 1:2 (w/w). Aliquots were removed at 10-min IV. Affinity chrornatog- 419 13.426 59.3 267
intervals and added to buffer containing 1 mg/ml BSA and soybean raphy on N46-
trypsin inhibitor. aminohexy1)-
The cleavage fragments resulting from the action of trypsin were oxamate-Seph-
analyzed on SDS-polyacrylamide gels. The trypsin to lactate dehy- arose
drogenase ratio was decreased to 1:50 (w/w) and the temperaturewas "From 50 g of frozen excised livers of LDH-BB/Bb.
1302 Lactate Dehydrogenase Allozymes

30 600

500
FIG. 1. Affinity chromatography
of the LDH-B, allozymes of F. het-
eroclitus. A, affinity chromatographyof 20 400
Step I1 material (68 ml, 3523 mg of pro-
tein, LDH-Ba/Bb) on a N'-AMP-Seph-
arose 4B column (2.5 X 20 cm; 6 pmol of 300
affinant/ml of gel) equilibrated with 20
mM sodium phosphate buffer (pH 7.00)
containing 1 mM 2-mercaptoethanol, 1 IO 200
mM EDTA, and 0.2 M NaCI. Fraction
volume was 20 ml and flow rate was 100
ml/h. The arrow indicates the addition IO0
of 0.25 mM NADH (20 ml) to the elution
buffer. The fractions with lactate dehy-
drogenase ( L D H ) activity which were
pooled as StepI11 material are indicated.
Absorbance readings (280 nm) were dis- Fraction Number
continuedafterapplication of there-
duced cofactor. B, affinity chromatogra-
phy of Step 111material (140 ml, 35.2 mg r

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I I I I I I I
of protein) on a N-(6-aminohexyl)ox-
amate-Sepharose 4B column (6 X 14 cm, B
10-11 pmol of affinant/ml of gel) equi-
librated with 20 mM sodium phosphate
buffer (pH 7.00) containing 1 mM
EDTA, 0.2 mM NADH, and 0.5 M NaCl. 10
The arrow indicatesthe removal of
NADH from the elutionbuffer. The frac-
tions indicated were pooled and concen-
trated by ultrafiltration on a Diaflo PM-
10 membrane. Absorbance readings a t
280 nm were made after blanking against
I
o
O8
06
the elutionbuffer. Readings were discon- N
4
tinued afterremoval of NADH. Theflow
rate was 200 ml/h and 20-ml fractions 04
were collected.

02

Fraction Number

andChrambach (15) thefrictionalretardation coefficient the 5% level (Xz3= 7.81).5

( K R )and the relative free mobility ( Yo)were obtained for Gel Filtration Behauior-The allozymes emerged as a single
each allozyme. A Ferguson (32) plot of the data is shown in peak from Sephadex G-200 columns with an apparent M , =
Fig. 2A. The patternof parallel lines isindicative of proteins 13.9 -+ 2.9 and a Stokes radiusof 4.44 f 0.03 nm. The specific
similar in molecular size (same KR),but different in electro- activity of the peak was found tobe reasonably constant (440
phoretic mobility (different Yo)(12). The 95% joint confi- f 40 units/mg).
dence ellipses for the two parameters are presented in Fig. Sedimentation Velocity Behavior-Both LDH-B; and
2B. LDH-Bk sedimented asa single symmetrical boundaryat two
Electrophoresis of the allozymes inSDS-polyacrylamide concentrations: 1.18 and 2.30 mg/ml. The corrected ( s ~ ~ . ~ )
gels a t two concentrations of acrylamide showed one major sedimentation coefficients were 7.21 f 0.085 and 7.42 f 0.095
band (>95%) with M , = 33.7 f 0.23. at 1.18 mg/ml, and 7.21 -+ 0.10 and 7.31 f 0.07 at 2.30 mg/ml
Isoelectric Focusing-Table I11 presents the PI estimates for LDH-B!j and LDH-B;, respectively; a partial specific vol-
for the LDH-B, allozymes. The observed PI difference (0.6 ume of 0.747 was used (26). These values compare favorably
pH unit) between the homotetramers (LDH-Bk versus LDH- with thevalue (7.22 f 0.27) found forLDH-Ba/Bhby isopycnic
B:) is consistent with a greater electrophoretic mobility for sucrose density centrifugation.
LDH-B!. LDH-Ba/Bh was resolved intothe expected five Extinction Coefficients-The molar extinction coefficients
major forms, with the heterotetramersdiffering in PI by 0.15 (AM) were the same for the LDH-B, allozymes. A value of
pH unit. The overall distribution of protein in LDH-Ba/Bh 2.05 k 0.08 x lo5 M" cm" was found assumingM , = 140,000.
based on the protein stain was 1.85:4.72:5.74:3.73:1.00 (Bi, The A280/A260 value was 1.55 f 0.14.
B$Bh, B;B& WB!, B!j) whereasfrom the activity stain the Specific Actiuity-The mean specific activity of the LDH-
proportions were 1.80:4.41:5.48:3.61:1.00.A goodness of fit -
test examining the departure from the expected proportions The subscript refers to the degrees of freedom available t.o the
(i.e. 1:4:6:4:1) gave XZ3= 5.99, which was not significant at statistic.
 Lactate
Allozymes
Dehydrogenase 1303
TABLEI11
TABLE
I1
Isoelectric points of the LDH-BI allozymes
A summary of the physiochemical propertiesof the LDH-B, allozymespH The
" gradient was attained in 5% a polyacrylamide
with 2% !v/
Value Method V) 3:1 mixture of pH 5-8 and 3.5-10 carrier Ampholines. Focuslng
Parameter (mean f S.E.)
-~ was done a t room temperature (-22 4 "C) for h. Protein was stained
Molecular mass (kDa) 187 k 10.2 Gel electrophoresis
with Coomassie brilliant blue R-250.
139 f.2.9 Gel filtration Isozyme Locality Genotype PI
147 f. 10.1 Gel filtration-sedimenta-
tion velocity LDH-B"B" Georgia LDH-B; 6.60 t 0.05
Subunit
(kDa)
mass 33.7 f 0.23 Sodium dodecyl sulfate gel Maryland 6.52 f 0.02
electrophoresis LDH-B"B~ LDH-B)
Maryland 7.20 f 0.03
Stokes radius (nm) 4.44 f 0.29 Gel filtration 7.07
LDH-BBB" f 0.03
Diffusion coefficient" 4.83 f 0.32 Gel filtration LDH-B$B; 6.90 f 0.02
(D20..,X 10" cmz/s)
LDH-BbBI 6.73 f 0.05
Sedimentation coefficient 7.26 f 0.16 Sedimentation velocity LDH-BP 6.60 f.0.03
(szo.w x 1 0 - 1 ~
S) LDH-B~B~ Maine LDH-B!i 7.20 f 0.05
AMa t 280 nm ( X M" 2.05 f 0.08 Spectrophotometry in 0.1 M Maryland _ _ 7.22 f 0.05
~
cm") (pHsodium phosphate
7.50) (microbiuret
method)
1.00
AZROIAZM 1.55 & 0.14
Specific activity (pmol/ 487 f 13.1 Spectrophotometry in 0.1 M
min/mg) (pH
sodium phosphate
7.50) containing 0.33 mM
pyruvate and 0.17 mM c
0 "

NADH E

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"~
IL
Calculated from the Stokes radius obtained with gel filtration by
a ~ o,50
Stokes-Einstein's formula. ._
>
._
c

9
-
0
c
._
c
0

I I I I I I I I I u
0
A L
l

160- - 0.00

;
I
- 45 40 50 55 60 65 70
0
2 Incubation Temperature
x P C1
- I40 -
FIG. 3. Heat inactivation profiles of the LDH-BI allozymes
- of F. heteroclitus. Purified lactate dehydrogenase was diluted in a
a
J
- 0.01 M sodium phosphate buffer containing 0.1% (w/v) BSA and 0.15
M NaCl. After heatingthe enzyme dilutions for 20 min at the
0
0 II designated temperatures, thesamples were cooled rapidly to 4 "C and
120 -
~

m assayedfor lactate dehydrogenaseactivity. 0, 0 LDH-B;; B, 0:

m LDH-Bi; A: LDH-B"/Bb.
- P
B4 allozymes was 487 f 13.1 units/mg.6 Variation between
IO0 I I I I I I I I I preparations varied by no more than 10%.
2 4 6 8

Stability Studies
The lactate dehydrogenase isozymes of many species are
known to have distinct susceptibilities to inactivationby heat
(33, 34), urea (35), and proteolytic enzymes (36). Generally,
the LDH-A, isozyme is more susceptible than LDH-B4 (37).
To test whether differences might exist between the LDH-B,
allozymes of F. heteroctitus, stability studieswere undertaken.
Heat Inactivation-Fig. 3 shows that the heat inactivation

tt 1
1
profiles for the LDH-B4 allozymes differ significantly. The
values for LDH-B;, LDH-B"/Bb, and LDH-B?were 57.5,
61, and 62.5 "C re~pectively.~
The standard errorof this estimate is:
01 I I I I
0 08 0 09 0 IO
K,
FIG. 2. A, Ferguson plot (32)of the electrophoretic mobility of the where A is the measured activity, B the protein concentration, anda
LDH-BI allozymes. The patternof parallel lines observedis expected and b their standard deviations, respectively. From several hundred
for "charge" isomers, that is, proteins that have different net charges, enzyme and proteinassays, the mean coefficient of variation of these
but the samemolecular size (12). B, The 95% joint confidence ellipses two measurements was approximately 2 and 5%, respectively. Hence,
for the KR and Yo estimates. Nonoverlappingellipses indicate a the coefficient of variation for thespecific activity was 5.3%.
nonidentity of the proteins at the 95% confidence level. The arrow ' The temperature a t which 50% of the enzyme activity remains
indicates a mean K R of 0.0905. I , LDH-Bg; 11, LDH-BBBb;III, LDH- after a specified period (e.g. 20 min) is designated the T w of that
B;Bb; IV, LDH-B"B2 and V, LDH-Bt. enzyme.
1304 Lactate Dehydrogenase Allozymes
To determine whether the addition of cofactor would sta-
bilize one allozyme more than another (34, 38), NAD+ was
added to the incubation mixture and the heat inactivation
procedure was repeated at each allozyme's Ts0.Thirty-three
per cent more activity remained for LDH-B,b in the presence
of 2 mM NAD' while a 30 and 25% increase was found for
LDH-Ba/Bb andLDH-B;, respectively. Although all the allo-
zymes were stabilized, nochange in the relativeorder of
stability was found in the presence of cofactor. Varying the
initial protein concentration 4- to 5-fold had no effect on the
heat stability of the allozymes.
The loss in activity due to heat inactivation can frequently
be described by a first order rate process (38). As shown in
Fig. 4, the heat inactivation kinetics of both LDH-B; and
LDH-B)I is consistent with a first order process. The results
further substantiate the dramatic difference in heat stability
between the allozymes. At 60 "C, the half-life of LDH-B; was
16 times shorter than that of LDH-Bi. The estimated ther-
modynamic activation parameters at 60 "C were AG$ (kcall
Time
(mi") mol) = 25.8 f 2.40 and 23.8 f 1.59, A H $ (kcal/mol) = 119 f
7.78 and 83.7 f 3.70, and AS* (cal/mol. "C) = 280 f 18.5 and
180 f 9.0 for LDH-B,b and LDH-B;, respectively.
The heat inactivation kineticsfor LDH-B"/Bb were not

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first order (Fig. 4B). This is not surprising since the pheno-
type contains an approximately equimolar ratio of the B" and
Bb subunits. Thesolid lines and symbols of Fig. 4B represent
the experimentalvalues, while the dashed lines are theoretical
curves assuming each subunit denatures independently (see
the figure legend for details). At 57.5 and 60 "C, LDH-B"/Bb
was significantlymore stablethan expected,whereas at
62.5 "C the theoretical and experimental curves were indistin-
guishable. At 65 "C, the rate of inactivation for LDH-B"/Bb
was identical with the rateobserved for LDH-B)I ( ts = 0.222).5
The heterozygous phenotype more strongly reflected the heat
resistance of the Bb-type subunit. This did notappearto
result from dissociation and reassociation of the heterotetra-
mers into the thermally more stable LDH-B)I homotetramers.
A mixture of the homotetramers was found to be inactivated
at exactly the expected rate for two independent processes
I I I I I I and no evidence of hybrid formation was observed when the
20 40 60
mixture was electrophoresed.
(min)
Time Urea Znactiuation-The loss in enzymaticactivity of lactate
dehydrogenase observed in urea solutions hasbeen attributed
to two phenomena: inhibition and inactivation (39). Urea has
1 I I 1 I I ' c been reported tobe a competitive inhibitor of pyruvate bind-
lP0
ing in teleost and elasmobranch (40), aswell as rabbit LDH-
A, (35). The inhibition is reversible at low urea concentrations
(35) while the inactivation is only partially irreversible and
apparently first order withrespect to time (39).
Above 0.5 M urea, the LDH-B, allozymes were inactivated
x by urea in a 10-min period. As with heat inactivation, we can
.-
c

.-> define a parameter, Cs0,which is the urea molarity at which

"
c

a 50% of the enzyme activity remains after incubation fora set

- 0.10
time (e.g. 10 min). In the absence of NADH, the G O values
0
c
.".
0
.- were 1.75 f 0.02, 1.50 f 0.03, and 1.24 f 0.03 M, while in the
E 0.05 presence of the reduced cofactor the values were 2.20 f 0.04,
U
2.10 f 0.04, and 2.00 f 0.02 M for LDH-Bi, LDH-Ba/Bb, and

experimental values found. The dashed line in B is the expected decay

67.5-C
rate assuming each subunit (B" and Bb) denatured independently;
I 1 I I I I that is, the fractional activity remaining at any one time is equal to
0 20 40 60
a simple sum of the decay rates of each homotetramer. This relation
Time
(min) can be written as: fractional activity = CbeCbL + C.e-kd where kh and
FIG. 4. Rates of heat inactivationof the LDH-B4 allozymes K. arefirst order rateconstants for LDH-B! andLDH-Biheat
of F. heteroclitus. A, LDH-Bk B, LDH-Ba/Bb; and C, LDH-BZ inactivation, respectively. The coefficients ch and c. are thefractional
The buffer used was 10 mM sodium phosphate (pH 7.50) containing proportions of the Bb and B". Based on the densitometry data, these
0.1% (w/v) BSA and 0.15 M NaCl. The solid symbols represent the values are taken as0.45 and 0.55, respectively.
 Lactate DehydrogenaseAllozymes
\ \
1305
LDH-B;, respectively. Therefore,theaddition of cofactor
stabilized all three allozymes to urea inactivation but there
was no change in the order of stability.
A measurable time dependence for urea inactivation was
observed with the LDH-B4allozymes only above 0.5 M. After
a n initial 5 to 10 min, the loss in activity followed first order
kinetics. Fig. 5 presents the observed time courses after cor-
1.67M
rection for the inhibition phenomena (39).Both homotetra-
mers exhibit first order kinetics with the more stable LDH-
B: allozyme inactivating 5 to 7 times slower than LDH-B; a t
urea concentrations of 1.33 to 1.67 M. As with heat inactiva-
tion, the time coursefor urea inactivation of LDH-Ba/Bb was
curvilinear. However, in contrast to heat inactivation, urea
inactivation of LDH-Ba/Bb could be adequately described by

I 1 I 1
'I
1.90M
I
assuming the B"- and Bb-type subunits inactivate independ-
ently (Fig. 5B).
IO 20 30 40 50 60 ProteolyticSusceptibility-The LDH-B4 allozymes were
tested for their sensitivity to three proteases (Table IV). In
Time (min)
response to treatment with trypsin and chymotrypsin, the
allozymes exhibited the same order of stability as found for
1.0 heat and urea inactivation: LDH-Bt > LDH-B"/Bb > LDH-
B;. Pronase treatment did not differentiate between the allo-
zymes.

Downloaded from http://www.jbc.org/ by guest on October 26, 2017

0
.-
c
.-
c Each allozyme had a distinct rateof inactivation by trypsin.
Based on a t test, the rate constants for trypsinolysis of LDH-
CK
0.5 B: and LDH-B; were significantly different (t4 = 14.7).
._
c However, addition of reduced cofactor (to 1 mM) stabilized
.-
c
w
both the enzymes sufficiently so that the rates of tryptic
8 inactivation were essentially identical for the homotetramers
( 4 = 1.19).
Jeckel et al. (41)and Millar (42)found that mild trypsino-

TABLE IV
Fractionul activity remaining for the LDH-B allozymes after
incubation wtih trypsin, chymotrypsin,and pronase
I I I I I 1 J Protease (25'C)
(0.005%, w/v) LDH B:
Allozyme
LDH-B'/Bb LDH-B:
IO 20 30 40 50 60
lime (min) Trypsin (30 min) 0.871 f 0.01 0.810 f 0.03 0.727 f 0.04
Chymotrypsin (30 min) 0.984 f 0.01 0.843 f 0.02 0.737 f 0.01
Pronase (10 min) 0.465 f 0.01 0.470 f 0.01 0.433 f 0.05

'i BSA-

Ovolbumtr

Chymolrypslnoqer
-0
-b

%,.J

1
-e

0.1 c
I-
I I
IO
I
20
I
30
Time (min
FIG. 5. Normalized inactivation kinetics for
-
40 50 60

the LDH-B,
-4

0 2 0 30 40 50 60 70 80 100
Tame ( m l n )
-f
-q

allozymes at various concentrations of urea. The activities are
expressed as fractions of the zero time values obtained by extrapola-
tion of linear first order plots: A, LDH-Bt (0);B, LDH-B"/Bb (A);
and C, LDH-B; (W). The solid lines for LDH-B! and LDH-B; are the
least squares regression lines, while the dotted curves for LDH-B"/Bb
are calculated as described in Fig. 4 using the rate constants deter-
mined for the homotetramers.
FIG. 6. An SDS-polyacrylamide gel (12.5% acrylamide) of
a tryptic digest of LDH-B; at 37 "C. The trypsin to lactate
dehydrogenase ratio was 1:50 (w/w). The first order rate constant
was estimated to be 1.34 f 0.32 X lo-' (rnin") by least squares
regression ( r = 0.998). Each channel was loaded with 10 gg of total
lactate dehydrogenase protein. Besides the standardsshown, aldolase,
soybean trypsin inhibitor, and ribonuclease A were electrophoresed.
1306 Allozymes
DehydrogenaseLactate
TABLEV turedtetramer.The absence of hybrid tetramers when a
Estimates of molecular masses of the tryptic fragmentsbased on mixture of the LDH-B4 allozymes is denatured is consistent
SDSpolyacrylamide gel electrophoresis with their conclusion.
The least squares line used to obtain the estimates was log M, = However, the structuralreorganization which occurs during
5.076 - 1.501RF; r = 0.997.
thermal inactivation is influenced by type of subunit contacts
limitsError
Component
Molecular
mass in the lactate dehydrogenase molecules. Bovine LDH-A, and
kDa B4 hybrids do not inactivate independently(33). Instead, the
a 34.3 31.1, 37.9 B-type subunitis more labile in an LDH-A2B2 hybrid than in
b 31.8 28.8, 35.2 the LDH-B4 homotetramer. There are two homologous and
C 21.2, 23.5 26.0
d 22.8 25.1 20.5, four heterologous interactions in LDH-A2B2, compared with
e 21.2 19.1, 23.5 six homologous interactions in the homotetramer. The LDH-
f 9.89 8.70, 11.2 B"/Bb allozyme which showed an increased stabilization of
g 8.61 7.52, 9.86 the B"-type subunit over that observed in the homotetramer
supports this observation. The heterologous interaction be-
lysis of lactate dehydrogenase generated a discrete size distri- tween the Ba/Bb subunits stabilized the B" subunit more than
bution of polypeptides. In order to examine whether a similar the homologous Ba/Ba contacts.
phenomenon could be observed for the LDH-B, allozymes, it The importance of stability differences between homolo-
was necessary to modify the incubation mixture. The trypsin gous and heterologous subunit contacts is further evident in
to LDH ratiowas reduced to 1:50 and the temperatureraised the sensitivity of lactate dehydrogenase to urea. Chan and
to 37 "C. Atselected intervals, aliquots were removed and Shanks (39),investigating the urea stability of bovine LDH-
subjected to SDS electrophoresis. Fig. 6 is a photograph of A/B hybrids, observed that theisozymes could be arranged in
the Coomassie-stained gel. The molecular mass estimates of an orderof decreasing stability: LDH-B, > LDH-A, > LDH-
the fragments and their 95% confidence limits are found in B3A > LDH-BA3 > LDH-BJA,. They interpreted this se-

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Table V. Three major size classes were found 31, 20-23, and quence asrepresentingthecontribution of freeenergy of
8-10 kDa. Theseareessentiallythesame size fragments interaction within the B and A subunit itself as well as the
observed for bovine and porcine lactate dehydrogenase (41, free energy of interactions between the subunits. The lower
42). stability of the hybrids was attributedtothenumber of
Phenylglyoxal Inactivation-Berghauser andFalderbaum homologous and heterologous subunit contacts possible in
(43) have found that porcine LDH-B4 andA, are inactivated each hybrid. LDH-B2/A2was thought to be less stable than
by phenylglyoxal through modification of a single arginine either LDH-B3Aor LDH-BA3because it potentially contains
residue apparently involved in substrate binding (43). The two homologous and four heterologous interactions, whereas
LDH-B4 allozymes were also inactivated by phenylglyoxal LDH-B3A or LDH-BA3containsthree of eachtype. The
withLDH-B;being inactivated significantly fasterthan heterologous subunit contacts were calculated to lower the
LDH-B! ( tI4= 9.91). Upon addition of reduced cofactor, both free energy of activation by 4.4 kcal/mol when substituted for
homotetramers were protected equally ( ts = 1.83). one set of the six homologous subunit interactions; thus, the
greater sensitivity of LDH-Bz/A2 to urea inactivation. A bi-
DISCUSSION nomial mixture of LDH-B, and LDH-A, hybrids inactivated
The Allozymes As Typical Vertebrate Lactate Dehydrogen- at amuch fasterratethanan equimolar mixture of the
ases-The typical vertebrate lactate dehydrogenase is a glob- homotetramers.
ular proteincomposed of four identical subunits ( M r= 35,000) With the LDH-Ba/Bb allozyme, however, the time course
with a molar extinction coefficient at 280 nm around 2.00 X of inactivation was the same as that expected assuming each
lo5 M" cm" (44). The sedimentation coefficients are in the isozyme in the heterozygous allozyme was inactivated inde-
range spo.uu = 7.2 to 7.7. The specific activityfor pyruvate pendently. This would indicate stability differenceswithin
reduction is usually around 360 units/mg with a slightly subunits themselves; otherwise, the behavior of LDH-Ba/Bb
highervaluefor lactate dehydrogenaseisolatedfrom cold- would resemble the bovine LDH-A/B binomial mixture.
blooded animals (45, 46). Based on the above criteria, the The free energy of activation at unit ureamolarity (39) for
LDH-B, allozymesfrom F. heteroclitus are typicalcold- LDH-B, equaled 24.4 5 0.37 kcal/mol, while the estimate for
blooded vertebrate lactate dehydrogenases. LDH-B; was 23.1 -+ 0.13 kcal/mol. Onekcal of additional
If one examinesthe differencesbetween the allozymes, energy was necessary to inactivate LDH-Biover that of LDH-
several interesting points emerge. The order of stability to B;.
the various denaturants was in each case LDH-B!i' > LDH- Consistent with the hypothesis that stability differences
B"/Bb > LDH-B;. Since enzyme stability is often correlated between theLDH-B, allozymes reside withinthesubunit
with adaptation temperature(47, 48), our results appear be to themselves are the results of inactivation by trypsin.
contradictory to an a priori prediction that the warm water Jeckel et al. (41) investigating the limited trypsinolysis of
allozymes would be most stable. What aspects of the lactate porcine LDH-A, and B, observed that concomitant with the
dehydrogenase protein structure are examinedby the various cleavage of peptidebondsthe activitydecreased andthe
inactivating agents? lactate dehydrogenase changed its conformation. Using NAD'
The initial effect of heat inactivation on lactate dehydro- and sulfite tostabilize the cleavage products, they were able
genase appears tobe restricted. Fondy et al. (33), investigating to show by SDS gel electrophoresis two new protein bands:
the temperature stability of bovine LDH-A, and B4 hybrids, one of M , = 22,000 and the other of 10,000. Treatment with
foundnochangeintheelectrophoreticpatternafter50% carboxypeptidase B was found torelease approximately 1 mol
inactivation. Jacobson and Braun (47) using differential scan- each of lysine and arginine/mol of subunit. Based on the
ning calorimetry to study the thermal denaturation of rabbit primary structureof porcine LDH-B, (48), the most probable
LDH-A,, observed only a single transition even though both site of cleavage liesin the region 101 to 115. This region
disassociation into subunits and loss of secondary structure contains the arginine-rich catalytic loop (residues 101, 109,
in the monomer units could occur. They concluded that the and 115). In the crystal structure of the apoenzyme, this
denatured state of lactate dehydrogenase may be the dena- highly charged region is completely accessible to the solvent
 DehydrogenaseLactate Allozymes 1307
(49, 50). However, intheternaryenzyme-NAD+pyruvate The basis for this statement comes from model experiments
complex, the loop is folded down onto the surface of the in which the degradation rates of proteins in bacterial and
molecule (50). mammalian cells were found to correlate with their sensitivity
Two residues in this region of particular catalytic impor- to various well characterized endopeptidases, such as trypsin
tance are arginine 101 and arginine 109. Arginine 109 inter- and chymotrypsin (Ref. 51 and references therein). Proteins
acts with the carboxyl group of the substrate, and arginine of greater sensitivity are degraded more rapidly within the
101 moves down to thevicinity of the pyrophosphatelinkage cell. In addition, proteins with low isoelectric points tend to
of the coenzyme. be degraded faster than those with neutral or basic isoelectric
Since the catalytic loop of lactate dehydrogenase has a points (52). LDH-B; has a lower PI (almost 0.6 pH unit) than
highly conserved primary sequence (47), mild trypsinolysis LDH-Bt. Sinceenzyme concentrations in uiuo are determined
like that describedabove should cleave Fundulus LDH-B4 by rates of both synthesis and degradation, and assuming that
allozymes a t Arg 101, 109, and 115yielding threepeptide the synthetic rate for allelic products is identical, the phys-
fragments differing in molecular weight by approximately 1 iological consequence of differences in degradative rate would
kDa. This is exactly what was found for the fragments in the be to lower in uiuo concentrations of LDH-B;. Tilley et al.
20-kDA class (Table V, Fig. 6). While the fragments with (53) useda similar argument to explain the higher serum
molecular masses around 10 kDa are also consistent with the glucose phosphate isomerase activity in humans possessing
cleavage in this region, those data must be interpreted cau- the more stable Singhallelic variant of that enzyme.
tiously because the relationship between log M , and R F ex- A problem for this hypothesis is the fateof LDH-Ba/Bh. If
hibits a considerable discontinuity at 10 kDa (18).The 32- the two homotetramers have different half lives i n uiuo, either
kDa fragment, which was not observed by Jeckel et al. (41), there must be a continuous dissociation and reassociation of
may represent cleavage at residue 23 (lysine) which is highly the molecules in the cell or the isozyme pattern of LDH-B"/
conserved for lactate dehydrogenases in which sequences are Bh must be nonbinomial. This is contrary to what has been
available (50). demonstrated i n vivo (54) and i n uitro (55) for lactate dehy-

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Although it is difficult to rule out an inactivating effect by drogenase isozymes; the lactate dehydrogenase tetramer does
cleavage at Lys 23, cleavage at Arg 101 and/or Arg 109 would not readily undergo reorganization.
involve removal of catalytically required residues. Assuming In summary, the LDH-B, allozymes of F. heteroclitusdiffer
the latter, then the differences in the rates of trypsinolysis significantly in their sensitivity to several structural pertur-
between the LDH-B4 allozymes would reflect a differential bants. When viewed as reflecting conformational differences,
accessibility of the loop region in the respective apoenzymes. the findings indicate that the northern cold water phenotype
LDH-B!, being the least susceptible to inactivation, may carry possesses a protein which is structurally more rigid than its
the loop more often to the surfaceof the enzyme than LDH- southern warm water counterpart.
B;. X-ray crystallography of lactate dehydrogenase (50) indi-
cates that cofactor binding triggers the withdrawalof the loop Acknowledgments-We thank Drs. G. Ackers, G. Somero, G.
region from the exterior which would make it less susceptible Greaney, S. Roseman, W.W. Cleland, and many others for useful
to trypsin. Since NADH causes only a 25% reduction in the comments on this manuscript. We thank Steve Gentry andStreamson
Chua for their able technical assistance. The artistic assistance of
rate constant forLDH-B; but almost a60%reduction for Dianne Powers and secretarialassistance of Diane Warr are also
LDH-B;, the loop region of LDH-B2 must be more accessible appreciated.
in the absence of NADH than LDH-Bk. This conclusion is
supported by the fact that both homotetramers have identical REFERENCES
inactivation rates in the presence of co-factor (i.e. NADH). 1. Wander, R. K. (1976) in Molecular Evolution (Ayala, F. J., ed)
Additional evidence for a differential exposure of the cata- pp. 21-45, Sinauer Associates Inc., Sunderland, MA
lytic loop between LDH-B; and LDH-Bk is the kinetics of 2. Powell, J. R. (1975) in Evolutionary Biology (Dobzhansky, T.,
phenylglyoxal inactivation. Berghauser and Falderbaum (43) Hecht, M. K., and Steer, W. C., eds) Vol. 8, pp. 79-119, Plenum
showed that modification of a singlearginine inporcine LDH- Press, New York
A4o r LDH-B4 resulted in complete inactivation of the enzyme. 3. Lewontin, R. C. (1974) The Genetic Basis of Evolutionary Change,
The inactivated enzyme retained the ability to bind the same 4. Columbia University Press, New York
Harris, H. (1976) Fed. Proc. 35, 2079-2082
amount of NADH as native enzyme. Although the specific 5. Kimura, M. (1979) Sci. Am. 241, 98-107
arginine residue was not identified, it is felt by some investi- 6. Place, A. R., and Powers, D. A. (1978) Biochem. Genet. 16, 577-
gators (50) that one of three following residues is involved: 59 1
Arg 101, Arg 109, or Arg 171. Because NADH did not influence 7. Powers, D. A,, and Place, A. R. (1978) Biochem. Genet. 16, 593-
the incorporationof radioactive phenylglyoxal,Arg 101, which 607
8. Place, A. R., and Powers, D. A. (1979) Proc. Natl. Acad. Sci. U.
is involved in charge neutralization of thepyrophosphate
S. A . 76, 2354-2358
linkage, is aless likely candidate than the other two(44). 9. Mosbach, K. (1974) Methods Enzymol. 34, Part B, 229-242
Whichever residue is modified, the observed rates of inacti- 10. Place, A. R., Powers, D. A., and Lee, Y. C. (1977) Anal. Biochem.
vation for the LDH-B4allozymes are consistent with a greater 83,636-647
exposure of the loop for LDH-B; than for LDH-Bk. The fact 11. Davis, B. J. (1964) A n n . N . Y. Acad. Sci. 121, 404-427
that the additionof NADH results in identical ratesof phen- 12. Hedrick, J. L., and Smith, A. J. (1968) Arch. Biochem. Biophys.
ylglyoxal inactivation is consistent with movement of the loop 126, 155-164
13. Markert, C. L., and Masui, Y. (1969) J. Exp. 2001.172, 121-146
closer or more often to the surface of the enzyme molecule 14. Fairbanks, G., Steck, T. L., and Wallach, D. F. H. (1971) Bio-
during cofactor binding. chemistry 10, 2606-2617
Physiological Significance of theStabilityDifferences- 15. Rodbard, D., and Chrambach, A. (1974) Electrophoresis and Iso-
Whether the differences in structural stability observed be- electric Focusing i n Polyacrylamide Gel (Allen, R., and Maurer,
tween the LDH-B4 allozymes are the consequence of func- H., eds) pp. 28-62, Walter de Gruyter, New York
tional differences is unclear. However, by exhibiting distinct 16. Laemmli, U. K. (1970) Nature (Lond.)227, 680-685
stabilities i n uitro, the possibility arises that the allozymes 17. Weber, K., and Osborn, M. (1969) J. Biol. Chem. 244,4406-4412
18. Neville, D. M. (1971) J . Biol. Chem. 246,6328-6334
may have distinct stabilities in uiuo; that is, one allozyme may 19. Wrigley, C. W. (1971) Methods Enzymol. 22, 4406-4412
be more susceptible to intracellular degradation than another.20. Righetti, P. G . , and Drysdale, J.W. (1976) in Laboratory Tech-
1308 Allozymes
Dehydrogenase Lactate
niques in Biochemistry and MolecularBiology (Work, 'I,. S., and 6163-6168
Work, E., eds) Vol. 5, Part 11, North-Holland/American Elsev- 40. Yancey, P. H., and Somero, G. N. (1978) J . Comp. Physiol. 125,
ier, New York 135-141
21. Vesterberg, O., and Svenson, H. (1966) Acta Chem. Scand. 20, 41. Jeckel, D., Anders, R., and Pfleiderer, G. (1973) Hoppe-Seyler's
820-834 Chem. Z. Physiol. 354, 735-748
22. Ackers, G . K. (1975) in The Proteins (Neurath, H., ed) Vol. 1, pp. 42. Millar, D. D. (1974) Biochim. Biophys. Acta 359, 152-176
1-94, Academic Press, New York 43. Berghauser, J., andFalderbaum, I. (1971) Hoppe-Seyler's Physiol.
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 Purification and characterization of the lactate dehydrogenase (LDH-B4)
allozymes of Fundulus heteroclitus.
A R Place and D A Powers
J. Biol. Chem. 1984, 259:1299-1308.

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